Citrate cross-feeding by Pseudomonas aeruginosa supports lasR mutant fitness

ABSTRACT Cross-feeding of metabolites between subpopulations can affect cell phenotypes and population-level behaviors. In chronic Pseudomonas aeruginosa lung infections, subpopulations with loss-of-function (LOF) mutations in the lasR gene are common. LasR, a transcription factor often described for its role in virulence factor expression, also impacts metabolism, which, in turn, affects interactions between LasR+ and LasR− genotypes. Prior transcriptomic analyses suggested that citrate, a metabolite secreted by many cell types, induces virulence factor production when both genotypes are together. An unbiased analysis of the intracellular metabolome revealed broad differences including higher levels of citrate in lasR LOF mutants. Citrate consumption by LasR− strains required the CbrAB two-component system, which relieves carbon catabolite repression and is elevated in lasR LOF mutants. Within mixed communities, the citrate-responsive two-component system TctED and its gene targets OpdH (porin) and TctABC (citrate transporter) that are predicted to be under catabolite repression control were induced and required for enhanced RhlR/I-dependent signaling, pyocyanin production, and fitness of LasR− strains. Citrate uptake by LasR− strains markedly increased pyocyanin production in co-culture with Staphylococcus aureus, which also secretes citrate and frequently co-infects with P. aeruginosa. This citrate-induced restoration of virulence factor production by LasR− strains in communities with diverse species or genotypes may offer an explanation for the contrast observed between the markedly deficient virulence factor production of LasR− strains in monocultures and their association with the most severe forms of cystic fibrosis lung infections. These studies highlight the impact of secreted metabolites in mixed microbial communities. IMPORTANCE Cross-feeding of metabolites can change community composition, structure, and function. Here, we unravel a cross-feeding mechanism between frequently co-observed isolate genotypes in chronic Pseudomonas aeruginosa lung infections. We illustrate an example of how clonally derived diversity in a microbial communication system enables intra- and inter-species cross-feeding. Citrate, a metabolite released by many cells including P. aeruginosa and Staphylococcus aureus, was differentially consumed between genotypes. Since these two pathogens frequently co-occur in the most severe cystic fibrosis lung infections, the cross-feeding-induced virulence factor expression and fitness described here between diverse genotypes exemplify how co-occurrence can facilitate the development of worse disease outcomes.


Fig. S1
Fig. S1 Citrate is the only tricarboxylic acid (TCA) cycle intermediate significantly enriched in LasR-cells across genetic background and media type.Volcano plots showing differential intracellular metabolite counts (log 2 LasR-/LasR+) for the laboratory isolates PA14 and ∆lasR on Artificial Sputum Medium (ASM) (A) or the LasR-clinical isolate compared to its closely related LasR+ progenitor on LB (B) relative to the −log 10 (P value).Metabolites are colored based on their categorization as dipeptides (lavender) or inclusion in the TCA cycle (light blue).4-hydroxy-2-heptylquinoline (HHQ) and N-butanoyl-L-homoserine lactone (C4HSL) which are known to be enriched and depleted in lasR mutants, respectively are also indicated.C4HSL was not significantly different in the ASM dataset between strains.C. The log 2 fold change and statistical significance of metabolites noted for LasR-over LasR+ strains in all three conditions tested (3 circles, left to right): PA14 ∆lasR / WT on LB, LasR-DH2415/ LasR+ DH2417 on LB, and ∆lasR / WT on artificial sputum medium (ASM).Circle fill is colored by relative log 2 fold change as indicated by scale ranging from -2 to 2. Significance as determined by Welch's t-test is indicated by circle outline (P value ≤ 0.05, solid outline and P value > 0.05 grey, dotted).Metabolites listed in grey text without numbers were not detected in our analysis.

Fig. S3
Fig. S3 TctED positively regulate citrate transport necessary for growth on citrate but not LB. A. Model illustrating the positive control both the sensor kinase TctE and response regulator TctD have on select genes involved in tricarboxylate transport (i.e.opdH and tctABC) in response to citrate.OpdH is a porin located in the outer membrane and TctABC is an inner membrane (IM) transporter.B.Growth in M63 base containing 10 mM citrate as a sole carbon source after 24 h from a single colony inoculum for the indicated strains.C. Quantification of PopdH -GFP -lacZ via beta-galactosidase assay for the indicated strains when grown for 24 h as colony biofilms on LB agar or LB agar supplemented with 20 mM citrate.D. Growth yield after 8 h subculture and exponential growth rate in 5 mL LB cultures.

Fig. S5
Fig. S5 In co-culture with wild type, ∆lasR cells exhibit increased RhlR-dependent rhlI promoter activity at the cellular level.A. Representative histogram of mKate2 negative counts by expression of green fluorescent protein (GFP), which was detected by FITC channel, of the ∆lasR PrhlI -GFP -lacZ cells in monoculture (light beige, solid line) or co-culture (darker beige, dotted line) with a wildtype strain expressing mKate2 constitutively under the Ptac promoter at a neutral site (illustrated in inset).Cell counts and median fluorescence intensity (MFI) indicated for cells lacking mKate expression (mKate O F F ) that exhibit GFP fluorescence (GFP O N ) after 6h subculture in LB.

Table S1
Relative intracellular metabolites detected for LasR+ and LasR-paired isolates on LB or Artificial Sputum Medium (ASM).a See attached excel file with raw and normalized metabolite counts with differential abundance and significance indicated. a

Table S2
Strains and plasmids used in this study b b See attached PDF file with strain identifier, description and source listed for each strain and plasmid.uld 2023 mBio Supplement Mould 2023 mBio Supplement Mould 2023 mBio Supplement Mould 2023 mBio Supplement Mould 2023 mBio Supplement Mould 2023 mBio Suppleme